Fluorescent Dyes

An Overview

A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescing agent. This can be a fluorescing protein – for example GFP – genetically linked to the protein of interest. If cloning is impossible – for instance in histologic samples – it is required to use other techniques like immunofluorescence staining to visualize the protein of interest. For this purpose antibodies are utilized, which are linked to distinct fluorescent dyes and bind to the adequate target structure either directly or indirectly. Moreover, with the help of fluorescent dyes fluorescence microscopy is not only restricted to proteins but it gives also the chance to stain nucleic acids, glycans and other structures. Even non biological substances like Calcium ions can be detected. This article provides an introduction to the commonly used fluorescent agents.

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Immunofluorescence

In fluorescence microscopy there are two ways to visualize your protein of interest. Either with the help of an intrinsic fluorescent signal – that means by cloning and therefore genetically linking a fluorescent protein to a target protein. Or with the help of fluorescently labeled antibodies that bind specifically to a protein of interest. For some biological questions it is more useful or even necessary to perform the latter one. In the case of histological samples, for example, it is not possible to use fluorescent proteins because in general the specimen is derived from an organism which does not hold any fluorescent proteins. Furthermore, if a functioning antibody is available, immunofluorescence is much faster than fluorescent protein techniques, were you have to clone the gene of interest and transfect DNA into the adequate cell. Another disadvantage of fluorescent proteins lies in their nature of being a protein themselves. With it, they have specific proteinous characteristics inside a cell which can lead to dysfunction or misinterpretations concerning the attached protein of interest. However, it should be considered that using fluorescent proteins is generally the method of choice to watch living cells.

Immunofluorescence makes use of the very specific binding affinity of an antibody to its antigen. This can have two different appearances. The easiest way is to use one fluorescently labeled antibody which is binding to the protein of interest. This is called direct immunofluorescence.

In most cases there are two forms of antibodies used. The first one binds to the target protein and is not fluorescently labeled itself (1st antibody). But a second one (2nd antibody) which is binding the 1st antibody specifically carries a fluorescent dye. This method is called indirect immunofluorescence and has several advantages. On the one hand there is an amplifying effect, because more than one 2nd antibody binds to one 1st antibody. On the other hand it is not necessary to label each antibody against your favorite protein all the time with a fluorescent dye but to use commercial fluorescently labeled 2nd antibodies. Broadly used fluorescent dyes for immunofluorescence are FITC, TRITC or several Alexa Fluor® dyes which are mentioned in the following.

FITC and TRITC

Fluorescein isothiocyanate (FITC) is an organic fluorescent dye which is still used in immunofluorescence and flowcytometry. It has an excitation/emission peak at 495/517 nm and can be coupled to distinct antibodies with the help of its reactive isothiocyanate group, which is binding to amino, sulfhydryl, imidazoyl, tyrosyl or carbonyl groups on proteins. Its basic form – Fluorescein – has a molar mass of 332 g/mol and is often used as a fluorescent tracer. FITC (389 g/mol) was one of the first dyes which was used for fluorescence microscopy and served as an origin for further fluorescent dyes like Alexa Fluor®488. Its fluorescence activity is due to its large conjugated aromatic electron system, which is excited by light in the blue spectrum.

Fig. 1: Drosophila embryo development, Green: FITC, Red: TRITC

A dye very often used in the same breath with FITC is its similar sounding partner TRITC (Tetramethylrhodamine-5-(and 6)-isothiocyanate). In contrast to FITC, TRITC is not a fluorescein but a derivate of the Rhodamine family. Rhodamines also have a large conjugated aromatic electron system, what leads to their fluorescent behavior. In contrast to FITC, TRITC (479 g/mol) is excited with light in the green spectrum with a maximum at 550 nm. Its emission maximum is lying at 573 nm. The bond to proteins (e.g. antibodies) is also based on a reactive isothiocyanate group.

Even if FITC and TRITC are still in use, they are rather weak fluorescent dyes and not recommended for state of the art microscopy. Their profit is based on their economical price.

Cyanines

This relatively small collection of fluorescent dyes was derived from cyanine which was also the origin for their names: Cy2, Cy3, Cy5 and Cy7. All of them can be linked to nucleic acids or proteins via their reactive groups. For proteinous labeling maleimide groups are used, for example. Interestingly – concerning fluorescence – Cy5 is sensitive to its electronic surrounding. This can be utilized for enzyme measurement. Conformational changes of the attached protein lead to positive or negative alterations in fluorescence emission. Furthermore Cy3 and Cy5 can be used for FRET experiments. Cyanine dyes are comparatively old fluorescent dyes and the basis for other fluorochromes with improved brightness, photostability, quantum yield etc.

Alexa Fluor® dyes

Alexa Fluor® dyes are a big group of negatively charged and hydrophilic fluorescent dyes which are used very often in fluorescence microscopy. Their appellation goes back to their inventor Richard Paul Haugland who named the dyes after his son Alex Haugland. The tag is a trademark of Molecular Probes (a subsidiary of life technologies). Furthermore the respective laser excitation wavelength is mentioned in their labeling. For example the very broadly used Alexa Fluor®488 has an excitation maximum at 493 nm, which allows excitation with a standard 488 nm laser. Alexa Fluor®488 has an emission maximum at 519 nm. With this characteristics, Alexa Fluor®488 has very similar properties to FITC. Although Alexa Fluor®488 is a fluorescein derivate, in contrast to FITC it has a better stability, brightness and lower pH sensitivity. All the Alexa Fluor® dyes are sulfonated forms of different basic fluorescent substances like fluorescein, coumarin, cyanine or rhodamin (e.g. Alexa Fluor®546, Alexa Fluor®633). Their molar mass ranges from 410 to 1,400 g/mol.

DNA staining

In fluorescence microscopy not only proteinaceous structures are of interest but also nucleic acids. Sometimes it is necessary to define the exact position or number of cells by the detection of their nucleus. One of the most common DNA stains is DAPI (4',6-diamidino-2-phenylindole) which binds to A-T rich regions of the DNA double helix. DAPI fluorescence intensity increases if attached to DNA compared to its unbound state. It is excited by UV-light with a maximum at 358 nm. Emission spectrum is broad and peaks at 461 nm. A weak fluorescence can also be detected for RNA binding. In this case, emission shifts to 500 nm. Interestingly, DAPI is able to permeate an intact plasma membrane. Therefore it can be used in fixed, as well as in living cells.

A second broadly used DNA stain option is the family of Hoechst dyes, which was originally produced by the chemical company Hoechst AG. Hoechst 33258, Hoechst 33342, and Hoechst 34580 are Bis-benzimides with intercalating tendency to A-T rich areas, whereupon the latter one is not used very often. Similar to DAPI they are excited by UV-light and have an emission maximum at 455 nm which is shifted to 510–540 nm in an unbound condition. Hoechst stains are also cell permeable and can therefore be used in fixed and living cells. A difference to DAPI is their lower toxicity.

A membrane impermeable DNA stain is Propidium-Iodide. With it, it is often used to differentiate between living and dead cells in a cell culture, because it can`t enter an intact cell. Propidium- Iodide is also an intercalating agent but with no binding preference for distinct bases. In the nucleic acid bound state, its excitation maximum is at 538 nm. Highest emission is at 617 nm. Unbound Propidium-Iodide excitation and emission maxima are shifted to lower wavelengths and lower intensity. It can also bind to RNA without changing its fluorescent characteristics. To distinguish DNA from RNA it is necessary to use adequate nucleases.

A dye which is capable to make a difference between DNA and RNA without previous manipulation is Acridine Orange. Its excitation/emission maximum pair is 502 nm/525 nm in the DNA bound version and turns to 460 nm/650 nm in the RNA bound state. Furthermore it is able to enter acidic compartments like lysosomes. There the cationic dye is protonated. In this acidic surrounding Acridin Orange is excited by light in the blue spectrum, whereas emission is strongest in the orange region. Because apoptotic cells have a lot of engulfed acidic compartments this makes it an often used marker for those kinds of cells.

In fluorescence microscopy it is often reasonable to stain cell compartments like lysosomes or endosomes and organelles like mitochondria. For this purpose there is a palette of specific dyes available which will be mentioned in this section.

One well known way to observe mitochondria is the utilization of MitoTracker®. This is a cell permeable dye with a mildly thiol-reactive chloromethyl moiety. With it, it can bind to matrix proteins covalently by reacting with free thiol groups of cysteine residues. MitoTracker® exists in different colours and modifications (s. Table 1) and is a trademark of Molecular Probes. In contrast to conventional mitochondria specific stains like rhodamine 123 or tetramethylrosamine, MitoTracker® is not washed out after destruction of the membrane potential with fixatives.

According to mitochondrial stains there are also dyes marking acidic compartments like lysosomes which are called LysoTracker. These are membrane permeable weak bases linked to a fluorophore. Most probably these bases have an affinity to acidic compartments because of protonation. LysoTrackers are also available in different colours (s. Table 1).

A comparable compartment to lysosomes is the vacuole in fungi like Saccharomyces cerevisiae. This membrane enclosed space is also of an acidic nature. One way to visualize it in fluorescence microscopy is the use of styryl based dyes like FM 4-64® or FM 5-95®.

When it comes to protein secretion experiments the Endoplasmic Reticulum (ER) is of a special interest. One classical dye to stain this compartment is DiOC6(3). It has a preference for the ER but still binds to other membranes like those of mitochondria. Another way to specifically stain the ER is to use ER-Trackers like ER-Tracker Green and Red. Both are BODIPY based dyes which are linked to glibenclamide – a sulfonylurease – which binds to ATP sensitive Potassium channels exclusively resident in the ER membrane. BODIPY (boron-dipyrromethene) describes a group of relatively pH insensitive dyes which are almost all water insoluble. This does not make them a very good tool for protein labeling but for lipid and membrane labeling.

The adjacent compartment to the ER – the Golgi appararatus – can be labeled with fluorescent ceramide analogs like NBD C6-ceramide and BODIPY FL C5-ceramide. Ceramides are Sphingolipids which are highly enriched in the Golgi apparatus.

With the help of further lipid based dyes it is possible to stain special membrane regions like lipid-rafts. These cholesterol rich domains can be visualized by using NBD-6 Cholestrol or NBP-12 Cholesterol amongst others (Avanti Polar Lipids).

Besides using special non-proteinacous fluorescent dyes to label cellular compartments it is also possible to stain the area of interest with the help of proteins with preferences for distinct locations in the cell. These proteins can be linked to a fluorescent dye and visualized in the fluorescent microscope. One example for such an approach is the usage of wheat germ agglutinin (WGA) which binds specifically to sialic acid and N-acetylglucosaminyl present in the plasma membrane. WGA is coupled to a fluorescent dye. With it the plasma membrane can be observed.

Ion imaging

In the case of neuronal studies, gene activity or cellular movement – for example – it is of interest to know about the ion concentration of the cell. Sodium, calcium, chloride or magnesium ions have a deep impact on many different cellular events. Typically, ions can be trapped with the help of fluorescently labeled chelators, which change their spectral properties when bound to the appropriate ions. This principle is realized in the calcium indicators fura-2, indo-1, fluo-3, fluo-4 and Calcium-Green, for example.

Interestingly there are also protein based calcium indicators. One of them is based on the jellyfish chemiluminescent protein aequorin. Interaction of aequorin, the luminophore coelenterazine, molecular oxygen and Ca2+ leads to the release of a blue light – a very prominent mechanism in the discovery of fluorescent proteins.

Fluorescent dyes and their excitation and emission wavelength peaks

All the dyes mentioned above are listed in the following table. Furthermore additional fluorescent dyes are mentioned together with their excitation and emission wavelength peaks.